Catalase Lab

Catalase Lab Activity
Enzymes are biological catalysts that help to carry out the thousands of chemical
reactions that occur in living cells. They are generally large proteins made up of several
hundred amino acids. In an enzyme-catalyzed reaction, the substance to be reacted,
the substrate, binds to the active site of the enzyme. The enzyme then converts the
substrate to products. Finally, the products are released into solution and the enzyme is
ready to help with another reaction. As is true of any catalyst, the enzyme is not used up
as it carries out the reaction but is recycled again and again. One enzyme molecule can
carry out thousands of reaction cycles every minute!!
Each enzyme is specific for a certain reaction because its amino acid sequence is
unique and causes it to have a unique three-dimensional structure. The "business" end
of the enzyme molecule, the active site, also has a specific shape. Because the active
site is so specific it will only bind with one kind of molecule.
An enzyme can be denatured (unfolded) by extreme heat, pH, or ionic concentration. If
this happens, the enzyme will no longer be functional because the shape of the active
site will be destroyed.
In this exercise you will study the enzyme catalase, which speeds up the breakdown of
hydrogen peroxide, H2O2, (a common waste product of cellular metabolism) into water
and oxygen.
The reaction is:
It’s important for cells to be able to break down hydrogen peroxide as soon as it’s
generated in the cell because hydrogen peroxide is quite toxic for cells. The products of
the reaction, water and oxygen, are not toxic for the cell. So catalase helps to protect
the cell from damage that could be caused by its own metabolic waste. Catalase is a
very important enzyme! Because it’s so important, catalase is found in many animal and
plant tissues.
Pre Lab Questions:
1. What is the reactant (substrate) in the reaction above?___________
2. What are the products in the reaction above?
3. Would this reaction happen by itself without the help of catalase?
PART A - Observe Normal Catalase Reaction
1. Place about 2 ml of the 3% hydrogen peroxide solution into a
clean test tube.
2. Using forceps and scissors cut a small piece of liver and add it
to the test tube. Push it into the hydrogen peroxide with a stirring
rod. Observe the bubbles.
What gas is being released? (consider the equation above—on
the first page) _____________________
Throughout this investigation you will estimate the rate of the
reaction (how rapidly the solution bubbles) on a scale of 0-5
(0=no reaction, 1=slow, ..... 5= very fast). Assume that the
reaction in step 2 proceeded at a rate of "4"
Assume that the normal reaction rate of catalase is a 3.
Recall that a reaction that absorbs heat is endothermic; a reaction that
gives off heat is exothermic. Feel the temperature of the test tube with
your hand.
Has it gotten warmer or colder ____________
Is the reaction endothermic or exothermic __________________
3. Pour off the liquid into a second test tube.
Assuming the reaction is complete, what is this liquid composed
of? (refer to the equation on page 1 again) _________________
What do you think would happen if you added more liver to this
liquid? ______________________
Test this (add more liver) and record the reaction rate. Reaction
Rate ___________ (1 – 5)
4. Add 2 more mL of hydrogen peroxide to the liver remaining in
the first test tube. What is the reaction rate? (1 – 5)______
Is catalase reusable? Explain how you know.
Part B - What Tissues Contain Catalase?
You will now test for the presence of catalase in tissues
other than liver. Place 2 ml of hydrogen peroxide in each of
3 clean test tubes and then add each of the three test
substances to the tubes. As you add each test substance, record
the reaction rate (0-5) for each tube in the data table.
Rate of Reaction (0-5)
Does tissue contain
Catalase?-YES OR NO
If YES in column 3,
how can you tell it
contains catalase?
PART C - What is the Effect of Temperature on
Catalase Activity?
1. Put a piece of liver into the bottom of a clean test
tube and cover it with a small amount of water. Place
this test tube in a boiling water bath for 5 minutes.
2. Remove the test tube from the hot water bath, allow
it to air cool, then pour out the water. Add 2 ml of
hydrogen peroxide.
CAUTION: Use a test-tube holder for hot test tubes.
What is the reaction rate for the boiled liver and peroxide? (O-5).
3. Put a small quantity of liver into 1 other clean test tube and 1 ml
H2O2. into another test tube. Put this test tube of liver and one of
H2O2 into an ice bath. After 3 minutes, pour the tube of H2O2 into
the corresponding tube of liver and observe the reaction
What is the reaction rate for the cold liver/peroxide? (O-5). _____
PART D - What is the Effect of pH
on Catalase Activity
1. Add 2 ml hydrogen peroxide to 3 clean
test tubes, then add:
Tube 1—add 1 ml acetic acid (vinegar) pH=2.6
Tube 2 – add 1 ml sodium bicarbonate solution (baking soda
solution) (base) pH = 9
Tube 3 – add 1 ml distilled water (neutral) pH = 7
Now add liver to each of the test tubes (try to do it all at about the
same time, so you can easily compare)
Rate of Reaction for: (0-5) Acid _____ Neutral _____ Base_____
Part E-What is the Effect of Salinity (high salt
conc.) on Catalase Activity.
1. Label a test tube “SP” for “Salty potato puree.” Use a
spatula to take A SMALL AMOUNT (about 5 chunks) of your
potato puree and transfer it into the “SP” test tube. Push it
down to the bottom of the test tube with a stirring rod.
2. Use a pipet to add 10 drops of salt solution to the SP test tube.
4. Use a pipet and measure 3ml of hydrogen peroxide. Place the
hydrogen peroxide into the “SP” test tube.
5. Record your observations on the data sheet provided.
Rate of Reaction for: (0-5) _________
1). Describe the relationship between catalase and hydrogen
peroxide. Indicate which is the enzyme, which is the substrate
and what occurs during the reaction.
2). Is catalase reusable? Use your data to support your answer.
3A). How does temperature and pH affect the reaction rate of
3B). Explain what happened to the catalase at the molecular
4). In 2-3 sentences, summarize the data and information
displayed in this graph.
5A). What were some independent variables in this lab?
5B). What was the dependent variable in this lab?
5C). List 3 constants:
5D). Positive Control=
Negative Control=
6). Why was it necessary to puree the plant tissue in order to test
for catalase activity?
7). Draw and label the following: enzyme catalase, the substrate
hydrogen peroxide and the active site. Also draw and label the
products formed from this reaction.
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